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Evolution of visual pigments
Exp. Eye Res. (1974) 18, 323-332
Evolution of Visual Pigments C. D. B. BRIDGES* Department of Ophthalwmlqy, New York New York, N.Y. 10016, U.S.A.
Medical Ceder,
AND
C. E. Xoranda
DELISLE
ResearchCentre, Pointe Claire, Quebec,Canada
Rod visual pigments exhibit a variety of absorption maxima, determined partly by the nature of the prosthetic group, retinal or 3-dehydroretinal, and partly by the apoprotein opsin. Some animal taxa exhibit little diversity. On the other hand, selective pressures arising from the different light habitats of aquatic environments are believed to have in fish visual pigments, which often produced the correspondingly wide range of A,,, extends over 80 nm. Opsins appear to have similar molecular weights. Latest estimates of molecular weight suggest that they have approximately 315 aminoacidresidues. Some species, notably members of the salmon&e, have visual pigments with different A,,, yet have diverged only during the past several million years. This would indicate a very high rate of protein evolution in circumstances where we should expect that amino acid substitution would be severely restricted. Certain fish species (e.g. the deepwater sculpin, Myoxocephulw) have mixtures of visual pigments with A,,, separated by only 6 nm. The physiological usefulness of such a pair is dubious, but it may indicate polymorphism at the gene locus coding for opsin. The use of retinol and 3-dehydmntinof;-the basis of rhodopsin and porphyropsin, may vary between quite closely related species. Extraneous factors such as light and hormones are known t#o be important in amphibian larvae and fishes, but there is also clear evidence for genetic influences, as revealed by the divergent evolution of the system in various populations of originally anadromous smelt after only 10 660 years of isolat’ion.
1. Introduction The varied aquatic light environments that fishes occupy have clearly exerted evolutionary pressure on their visual pigments. More than 25 000 000 years ago the argentines and salmonidsdiverged from each other. Many of the former group became deep-seafishes and evolved visual pigments displaced towards the blue, so matching the blue-shifted spectrum of ambient light available for vision. Most of the salmonidae invaded fresh waters from the sea,either permanently or during spawning migrations, and hence developed red-shifted visual pigments corresponding to the reddish light that is often a feature of the freshwater environment (for review, seeBridges, 1972). The spectral location of a visual pigment absorption band can be influenced in two ways. One is to modify the apoprotein (opsin) while retaining the sameprosthetic group, viz. retinal in a rhodopsin or 3-dehydroretinal in a porphyropsin. Alternatively, rhodopsins and porphyropsins can be interchanged by substitution of one prosthetic group for the other, the apoprotein remaining unaltered. While many such changes have been accomplished by speciesevolving in a distant past under environmental conditions that are ill-defined from the standpoint of spectral light distribution, apparently somehave been more recent, occurring within the last few million years. The dominant influence of this period, which affected both speciation and distribution of many of the fishes discussedhere, was the advance and retreat of the great icesheetsduring the glacial epochs of the Pleistocene era, the last of which reached its maximum only 17 000-20 000 years ago.
2. Methods and Materials Collection
of material
Whole eyes or retinas were always obtained in dim red light from fish that) had been dark-adapted for 2-3 hr while still alive. Extracts of material from fish that were dead or nearly dead when placed in the dark frequently yielded some ,iso-pigment (9.cis prosthetic group) mixed with their natural visual pigment (the observations of Rotmans, Daemen and Bonting, 1972 and Putterman and Rollins, 1973 on regeneration of visual pigments may have some bearing on this finding). The ocular tissues were placed in stoppered vials, one for each fish (the retinas were immersed in &r/10 pH 7 phosphate buffer), sealed in light-tight containers and frozen on dry ice for transport to New York where they were transferred to a deep freeze until they were processed (usually within 1 week of collection). Extraction
procedure
Retinas were extracted according to the method of Bridges and Yoshikami (197Oa). Each pair of whole eyes was placed in a Petri dish containing 2.5 ml of McIlvaine’s pH 4% phosphate-citrate buffer and the choroid and retina scraped away from the sclera. After the sclera and lens had been picked out with forceps, the buffered suspension of the remaining tissues was pipetted into a 3-ml polypropylene centrifuge tube and centrifuged for 15 min at 17 000 rev/min in the 85-34 rotor of a Sorvall RC-2B centrifuge. This method was used because the retina became friable and extremely difficult to dissect out quantitatively after freezing and thawing. After centrifuging, the sedimented tissue was washed twice with distilled water then extracted with 2% digitonin (Merck reagent, thoroughly boiled before use to ensure maximum stability). The impurity of such extracts did not hinder the subsequent measurement of visual pigment composition. Similar results were obtained on occasions when comparatively pure preparations were made by first suspending the ground ocular material (without sclera and lens) in 37.5% sucrose, diluting, centrifuging and then washing several times with distilled water before extracting with digitonin. Analysis
and measurement
of viswll
pigment
composition
Visual pigments (in 2% digitonin solution containing 10 mM-hydroxylamine at pH 8.5) were identified by bleaching them in successive stages with monochromatic lights. The technique used was essentially that described by Dartnall (1952). The percentage of rhodopsin and porphyropsin was determined as outlined by Bridges and Yoshikami (1970b). A Zeiss PM& II spectrophotometer was used for measurement of absorption spectra, and bleaching was effected either by exposure to radiation from a high-intensity Bausch and Lomb monochromator in conjunction with suitable stray light filters, or to the non-isomerizing light from an incandescent lamp filtered through a yellow Corning glass no. 3-72. Liver “vitamins
A”
The methods used for analysis of the percentages of retinol and 3-dehydroretinol stored in the livers were identical with those described by Bridges and Yoshikami (1970a). A Cary 14 spectrophotometer was used to measure absorption spectra.
3. Results and Discussion Recent emergence of different
rhodopsins
In the salmonid fishes there changes that apparently involve
in the salmon&
are numerous examples of recent visual pigment the opsin moiety. Thus the rhodopsin of all Salmo
EVOLUTION
OF
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325
PIGMEN’TS
species examined (Munz and Beatty, 1965) has an absorption maximum at 503 nm and is quite distinct from that of Salve&us nama&ush, where the absorption maximum is at 511-512 nm (Munz and McFarland, 1965; Bridges and Delisle, 1974). It is considered unlikely that these genera diverged more than two million years ago (see Norden, 1961). Differences between rhodopsins of species within the genus Salve&us must, have occurred even more recently: the absorption maximum may be at, 51 l-512 nm for the lake charr (Salvelinus namaycush), at 508 nm in the Arct,ic charr and its derivatives (S. alp&us), or at 503 nm in the brook charr (S. fontinalisz Munz and McFarland, 1965; Bridges and Yoshikami, 1970a; Bridges and Delisle, unpublished). Similarly, while in the genus Coregonusthe rhodopsin is usually about 510 nm (Bridges and Yoshikami, 1970a) one speciesthat diverged in the British Isles no more than 20 000 years ago (Maitland, 1970) has a rhodopsin with absorption maximum shifted some 10 nm towards the red end of the spectrum (Bridges, 1965). How do such changescome about? Some evidence suggeststhat vertebrate visual pigments are homologous proteins (Heller, 1969) containing about 315 amino acid residues (a value based on the molecular weights of Robinson, Gordon-Walker and Bownds, 1972; Heitzman, 1972 and Daemen, de Grip and Jansen, 1972). Evolution by a.mino a.cid substitution in these proteins, however, would be severely restricted by the necessity for preserving certain essential features of the steric conformation at, the chromophore site (Bridges and Yoshikami, 1970a) and possibly by other considerations arising from the fact that rhodopsin is an important integral part of the photoreceptor outer segment membranes. The less rigorously defined fibrinopeptides show the highest mutation rates, amounting to 90 accepted point mutations or 52 observed differences per 100 amino acid residues-over 100 000 000 years (Dayhoff, 1972). In a visual pigment, this would mean a maximum observable difference between t’wo divergent lines of only 6.4 amino acids after 2 000 000 years. It is conceivable, of course, that new visual pigments could emerge quite rapidly if they were determined by a seriesof dominant and recessive alleles. In the salmonidae, however. codominance was established when S. namaycush x S. fontinabis hybrids were found to have mixtures of the parent pigments, the rhodopsins being separated by 8-9 nm (McFarland and Munz, 1965). Although such mixtures are extremely difficult to detect, in retinal extracts, in the following instance we document a thorough investigat)ion of the visual pigment from a glacial relict specieswhich we conclude possesses two rhodopsins separated by only 6-7 nm. Two closely si&ar rhodopsins ,in ~G4nE pigment evolutionZ
in the ret&a
of the deep-water
sculpin--rc
possible
stage
Illustrated in Fig. 1 are the absorption spectra from a retinal extract of the deepwater sculpin (Myoxocephalus quadricornis thompsonii) netted in a glacial lake in the Ottawa, valley, Quebec. It was first reported there by Delisle and van Vliet (1968). The absorption maximum is at 511 nm, and an initial red irradiation yielded a reckdisplaced difl’erence spectrum with maximum at 514-515 nm. This observation is often encountered in extracts which have mainly rhodopsin contaminated with a t*race of porphyropsin, such as those from Coreyonus &e&i. As shown in Fig. ‘L, however, in these casessubsequent difference spectra are identical with each other becausethe initial irradiation had creamed off the trace pigment. But in the caseof the seulpin, subsequent spectra shifted progressively as bleaching proceeded until a final value of 508-509 nm was reached. Even when the tirst irradiation bleached only 5%, of t,hetotal pigment (Fig. l), the corresponding difference spectra still had maxima
Wovelength
(nm)
FIN. 1. The problem of Myoxocep?dus visual pigments. The initial irradiation with X 660 nm light bleached only 5% of the total pigment: this was followed by irradiation at 650, 640 and 620 nm which left only 6% of the original pigment. This was then bleached by a final “yellow” (incandescent lamp filtered through Corning no. 3-72). In these experiments the difference spectra moved progressively 514-515 to 508-509 nm, as illustrated in Fig. 2. Temperature 25”C, hydroxylamine 10 mM, from h,,, pH 8.5, digitonin 2%.
c xl 100 P x t5 0” a
I
I
I
I,
400
I
III,,,,
I,,,
500 Wavelength
600 (nml
2. Difference spectra. (a) An example (from the retina of the whitefish Coregonus artedii) of a rhodopsin “contaminated” with a few per cent of porphyropsin. The initial red irradiation removes this pigment and produces a difference spectrum (dotted) that is displaced relatively to all subsequent ones (+ , x , A), which are identical with each other. These curves represent the bleaching of the purified rhodopsin, which has its absorption maximum at 510-511 nm. (b) The range of ourves obtained during the experiment in Fig. 1, where the visual pigment from Myoxocephalus retina was analyzed. In this case the spectra shift progressively from X,,, 5145 to 08 nm (. . ., the overall difference spectrum obtained by subtraction of the final fully bleached spectrum from the first). FIG.
EVOLUTION
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PIGMENTS
447
at 514-515 nm. Similarly, the shift was not due to a trace of blue-sensitive pigment, for a yellow irradiation following a red bleach that had removed all but 6% of the total pigment still produced a difference spectrum with maximum at 50&509 nm. The range of difference spectra obtained in a typical experiment on an extract from a single fish is illustrated in Fig. 2. Since all extracts were at 25°C and contained 10 m;\l-hydroxylamine it is considered very improbable that these observations are a,ttributable to photoproduct interference. Moreover, the initial spectra do not show the narrowing effect that would result from a red-displacement due to the formation of a substance with an absorption band overlapping that of the parent pigment. The most plausible conclusion, therefore, is that, these extracts contain roughly equal amounts of two rhodopsins with absorption maxima close t*o 514 and 508 nm respectively. The aqlvantage of two such pigments, in visual terms: is dubious. Since both pigments are present in individual specimens (although it is not known whether they are in the same retinal photoreceptors), it is possible that, this is evidence for gene duplication and subsequent mutation, perhaps after the species entered freshwaters from circumpolar seas a,t a time prior to the Wisconsin glaciation (McAllister, 1959). Post!yl&cd
evolution
of rhodopsin
ad
porphyropsi,l
i,~ isolated populations of thr
smelt (Osmeruseperlanus) We turn now to a casewhere in the course of evolutionary change the prosthet#ic group has been switched around. Because the porphyropsins as a group absorb at longer wavelengths than the rhodopsins they are more prevalent in freshwater fish retinas. Many diadromous speciessuch as the Pacific salmon and lamprey exchange their marine rhodopsin for porphyropsin at the onset of their spawning ascent into freshwat,ers (Crescitelli, 1956; Wald, 1957; Beatty, 1966). Consequently, it might be exprct’ed that if such a speciesbecame permanently landlocked. perhaps becauseof chaugesin climate and physiography of a region, there would be selective pressureto retain t’he red-sensibive pigment. In order to learn more about the recent evolution
100 miles
IJIG. 3. Sites where various smelt samples were taken (methods: scoop net, gill net or rod-and-line). (1) Rimouski: (2) St Lawrence, Rivibre Boyer; (3) St Lawrence, St Nicolas: (4) L. Mandeville; (5) L. Ouimet: (6) L. Heney; (7) L. Mesch; (8) L. Memphremagog; (9) L. Massawippi; (10) L. Utopia, Pl’ew Brunswick. Arrows indicate transplantations.
3’8
c’. D.
B.
BRIDGE,S
ASD
c’. E.
DELISLE
of this system, we have studied the distribution of rhodopsin and porphyrophin in different populations of the smelt, Osmerus eperlanus, a normally anadromous species that became landlocked in the St Lawrence Lowlands in Quebec during the retrea,t of the ice at the close of the last (Wisconsin) glacial epoch. Since isolation t,he migratory habit has persisted, perforce restricted to the ascent of small feeder streams. The locations of our various capture sites are marked on the map in Fig. 3. As might have been anticipated, the “free” smelt taken during their upstream spawning migration from the St Lawrence River (Osmerus eperlanus modax) a,nclt,he River Conway in the British Isles (Osmerus eperlanus e~erlanus) possessedvisual pigment composedalmost entirely of porphyropsin (absorption maximum 542 z 1 nm). The corresponding rhodopsin with absorption maximum at 512 nm predominated in non-spawners taken later in the year at site 1, near the St Lawrence estuary. Pure porphyropsin was also found in spawning smelt from the lake at site 4 (separated from the river by only 20 miles) and in Lake Utopia (site 10) near the Atlantic coast, where isolation occurred during upwarping of the landmass as the weight of glacier ice was removed. TABLE
1
Distribution of rhodopsin and porphyropsin in various spawning populations of the smelt (Osmerus eperlanus)
Source
L. Heney L. L. L. L. L. L. St R.
(6)
Comments
*
giants dwarfs
Utopia (10) Meach (7) Ouimet (5) Memphremagog (8) Maseawippi (9) Mandeville (4) Lawrence R. (R. Boyer) Conway (Gt. Britain)
Visual pigment composition i Mainly Mainly Gr0S.s porphyropsin rhodopsin mixture (more than (O-70% (more than porphyropsin) go(,):) 90%)
sympatrio
(2)
races
introd. introd.
from from
“free” “free”
fish fish
site 10 site 7
* Numbers in parenthesis refer to sites depicted on the map in Fig. 3. Two sites (1 and 3) were sources of non-spawning fish only. t Extracts were always prepared from individual fish. $ As in trout and rudd (Reuter, White and Wald, 1971; Muntz and Northmore, 1971), the dorsal half of the retina usually had more rhodopsin than the ventral, the maximum difference amounting to nearly 30%. The eyes of L. Massawippi specimens were not divided.
Contrary to expectation, however, it soon became apparent that smelt taken from other isolated lakes, although taxonomically indistinguishable from those discussed above, had very different visual pigment compositions. These are summarized in Table I. High proportions of rhodopsin were found in spawning fishesfrom sit,es8 and 9 on the southern side of the St Lawrence Lowlands. In fact, some individual specimens lacked porphyropsin altogether, although others ranged up to 60%. Over a period of
EVOLUTION
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PIGMENTS
329
3 years the average pigment composition of spawning fish at site 8 did not change significantly-there was 19% porphyropsin in 1971, 24% in 1972 and 26% in 1973. Porphyropsin had almost completely vanished from the eyes of smelt collected further westwards at site 6, in the Gatineau north of Ottawa. This lake supports a giant and dwarf race of smelt (Delisle and Veilleux, 1969), which are reproductively isola.ted by virtue of their different spawning times. In this instance, neither was found to have more than a few per cent of porphyropsin, either during spawning or at, any other time. Even the young had pure rhodopsin, irrespective of whether they were collected in summer a few months after hatching, or through holes in the ice at the end of the following winter. We then went on to examine fish from two other lakes (5 and 7) in the region, respectively north and east of site 6. Fifty years ago the lake at site 7 had been stocked by transplantation of fertilized eggs from Lake Utopia (lo), nearly 500 miles away. Subsequently, there was a second transplantation from site 7 into the lake at site 5. Both transplanted colonies differed from those of site 6 but were identical with the parent population in having nearly pure porphyropsin during spawning. From the point of view of light environment, these various lakes appear to differ littlta from each other (with the possible exception of the very shallow lake at site 4). so the blue-shifted visual sensitivity that would be associated with a predominantly rhodopsin system is not manifestly adaptive. What is the basis for these differences between smelt populations? Although vera high doses of exogenous preformed 3-dehydroretinol can effect a porphyropsin increase in kokanee salmon and rainbow trout (Beatty, 1972; Jaquest and Beatty, 1972), a converse effect attributable to lack of this precursor does not explain the situation in smelt. Thus the rhodopsin-rich site 6 group had as much as 500/, of their liver retinols in the form of 3-dehydroretinol: in contrast, the porphyropsin-rich group at site 5 had only 15%. The striking difference between the pigments of the autochthonous fish of site 6 and the transplanted populations of sites 5 and 7 suggest that local conditions such as feeding, predation and light-environment are probably not factors that determine visual pigment balance in the smelt, even though their importance in other species has been established (Dartnall, Lander and Munz, 1961: Bridges, 1965; Bridges and Yoshikami, 1970b; Allen, 1971). We conclude, therefore. that, we are dealing with different races of smelt where the ability to synthesize porphyropsin is genetically determined. Opsins do not appear to be selective for retinal or 3-dehydroretinal (cattle opsin will accept indiscriminately the correct &-isomers of retinal and 3-dehydroretinal to form t(he parent rhodopsin or its porphyropsin counterpart; Wald, 1953). Consequently. degeneration of the porphyropsin system may arise from failure of the ocular tissue?: t,o sequester preformed 3-dehydroretinol (originating in the liver) or from loss of any enzymes required specifically for utilization of 3-dehydroretinol. Possibilities include the ester hydrolase, alcohol dehydrogenase (unlikely) or the isomerizing system. Alt)ernatively, ocular 3-dehydroretinol may originate de novo by dehydrogenation of retinol, perhaps within the pigment epithelium* (cf. Ohtsu, Naito and Wilt, 1964; Naito and Wilt, 1962; see also Bridges and Yoshikami, 197Oc). Such a process would requirr a 3,4-dehydrogenase, or possibly a series of isoenzymes of varying efficacies * In this connection it is worth noting that the pigment epithelium of the goldfish contains onl) 3-dehydroretinol, even after massive injections of retinol (Bridges, unpublished), indicating that the tissrle accumulates 3.dehydroretinol preferentially (it is present in the liver) or that it dehydrogenates retinal before the latter reaches detectable proportions.
330
L’. D.
B.
BRIDGES
AKD
C. E.
DELISLE
(the salmonids typically maintain multiple molecular forms of many enz~n~t+~ probably the result of extensive gene duplication; Massaro, 1972). This would piovitle the genetic basis for individual variation. The time available for evolution of these different races of smelt is determined 1)~ the chronology of the Champlain Sea episode, a marine inundation of the St Lawrence Lowlands that followed in the wake of the retreating Wisconsin ice. The fishes of sites 6, 8 and 9 probably became isolated from the main gene pool of the “free” river smelt while the Sea was shoaling and freshening at its terminal Lampsilis La,ke phase. This would place the date between 11 000 and 8400 years ago (Prest. 1970). ACKNOWLEDGMENTS
This work was supported by PHS Research Grant no. 5 ROl EY 00461 from the National Eye Institute. Throughout the major period of the project, Dr Delisle was affiliated to the Aquarium de Mont&al. We gratefully of Dr D. D. Beatty and ,Mr Roy Bradford in collecting New Brunswick.
acknowledge the valuable assistance eyes from the smelt of Lake Ut,opia,
REFERENCES Allen,
D. M. (1971).
Photic
control
of the proportions
of two
visual
pigments
in a fish.
T;isior~ ties.
11, 1077. Beatty, D. D. (1966). A study of the succession of visual pigments in Pacific salmon (Oncorhyncus). Cad. J. Zool. 44,429. Beatty, D. D. (1972). Visual pigment changes in salmonid fishes in response to exogenous Lthyroxine, bovine TSH and 3-dehydroretinol. Vision Res. 12, 1947. Bridges, C. D. B. (1965). Absorption properties, interconversions and environmental adaptation of pigments from fish photoreceptors. Cold Spring Harbor Symp. Quant. Biol. 30,317. Bridges, C. D. B. (1972). The rhodopsin-porphyropsin visual system. In Handbook of Sensory Physiology, vol. viii (l), pp. 417480. Springer-Verlag, Berlin. Bridges, C. D. B. and Delisle, C. E. (1974). Brief observations concerning the visual pigments of some selected fishes from Lake Heney, Quebec, a relict of glacial Lake Gatineau. Vision Res. 14, 187. Bridges, C. D. B. and Yoshikami, S. (1970a). Distribution and evolution of visual pigments in salmonid fishes. Vision Res. 10, 609. Bridges, C. D. B. and Yoshikami, S. (197Ob). The rhodopsin-porphyropsin system in freshwater fishes 1. Effects of age and photic environment. Vision Res. 10, 1315. Bridges, C. D. B. and Yoshikami, S. (197Oc). The rhodopsit-porphyropsin in freshwater fishes 2. Turnover and interconversion of visual pigment prosthetic groups in light and darknessrole of the pigment epithelium. r&ion Res. 10, 1333. Crescitelli, F. (1956). The nature of the lamprey visual pigment. J. Gen. Physiol. 39,423. Daemen, F. J. M., De Grip, W. J. and Jansen, P. A. A. (1972). Biochemical aspects of bhe visual process XX. The molecular weight of rhodopsin. Biochim. Biophys. Acta 271,419. Dartnall, H. J. A. (1952). Visual pigment 467, a photosensitive pigment present in tenth retinae. J. Physiol. 116,257. Dartnall, H. J. A., Lander, M. R. and Munz, F. W. (1961). Periodic changes in the visual pigment of a fish. In Progress in Photobiology (ed. Christensen, B. and Buchmann, B.). Pp. 203-213. Elsevier, Amsterdam. Dayhoff, M. 0. (1972). Atlas of protein sequence and structure. National Biomedical Res. Foundation, Washington, D.C. Delisle, C. and Van Vliet, W. (1968). First records of the sculpins Myoxocephalus thompsonii and Cottus ricei from the Ottawa valley, southwestern Quebec. J. Fish. Res. Bd. Canada 25,273;~. Delisle, C. and Veilleux, C. (1969). R&partition geographique de l’eperlan arc-en-ciel Osnrerus eperlanus mcrdax et de Glugea hertwigi (sporozoa: microsporidia) en eau deuce, au Qn6ben. Natural&e Can. 96, 337. Futterman, S. and Rollins, M. H. (1973). Evidence for the involvement of a reduced flavin isomerization catalyst in the regeneration of bleached rhodopsin. Invest. Ophthalmol. 12, 234.
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Heitzman, H. (1972). Rhodopsin is the predominant protein of rod outer segment membranes. Nature New Biol. 235, 114. Reller, J. (1969). Comparative study of a membrane protein. Characterization of bovine, rat and frog visual pigments. Biochem. 8, 675. Jaquest, W. L. and Beatty, D. D. (1972). Visual pigment changes in the rainbow trout, S&LO guirdnerii. Canad. J. Zool. 50, 1117. Maitland, P. S. (1970). The origin and present dist,ribution of Coregonus in the British Isles. In Biology of Coregonid Fishes (Ed. Lindsey, C. C. and Woods, C. 8.). Pp. 99-114. University of Manitoba Press, Winnipeg. Massaro, E. J. (1972). Isozyme patterns of coregonine fishes: evidence for multiple cistrons for lactate and malate dehydrogenases and achromatic bands in the tissues of Prosobium cylindraceum (Pallas) and P. coulteri (Eigenmann and Eigenmann). J. Exp. Zool. 179, 247. Mc,4llister, D. E. (1959). The origin andstatus of the deepwater sculpin. iWyoxocepha,Zua thompsonii. a Nearctic glacial relict. Bull. Nat. Mus. Canada 172, 44. McFarland, W. N. and Munz, F. W. (1965). Codominance of visual pigments in hybrid fishes. Science (New York) 150, 1055. Muntz, W. R. A. and Northmore, D. P. M. (1971). Visual pigments from different parts of the retina in rudd and trout. Vision Res. 11, 551. Munz. F. W. and Beatty, D. D. (1965). A critical analysis of the visual pigments of salmon and tsout. Vision Res. 5, 1. Munz, F. W. and McFarland, W. N. (1965). B suggested hereditary mechanism for visual pigments of chars (Salvelinus spp.). Nature (London) 206,955. Naito, K. and Wilt, F. H. (1962). The conversion of vitamin A, to retinene, in a freshwat,er fish. J. Biol. Chem. 231, 3060. Norden, C. R. (1961). Comparative osteology of representative salmonid fishes, with particular reference to the grayling (Thyrnallus arcticus) and its phylogeny. J. Fish. Res. Bd. Cnnndn 18, 679. Ohtsu, K., Naito, K. and Wilt, F. H. (1964). Metabolic basis of visual pigment conversion in metamorphosing Rana catesbiana. Develop. Biol. 10, 216. Prest . V. K. (1970). Quaternary geology of Canada. In Geology and Economic Xinera.1.r of Canadrr, 5th ed. Econ. Rep. 1, chap. xii, pp. 676-725. Department’ Energy, Mines and Resources. Ottawa. Reuter, T. E., Whit,e, R’. H. and Wald, CT, (1971). Rhodopsin and porphyropsin fields in the adult, bullfrog retina. J. Gen. Physiol. 58, 351. Robinson, W. E., Gordon-Walker, A. and Bownds, D. (1972). Molecular weight of frog rhodopsin. Xature New Bill. 235, 112. Rotmans, J. P., Daemen, F. J. M. and Bonting, S. L. (1972). Biochemical aspects of the visual process XIX. Formation of isorhodopsin from photolyzed rhodopsin by bacterial action. Biochim. Biophys. Acta 267,583. Wald, G. (1953). Vision. Fed. Proc. 12, 606. Wald, G. (1957). The metamorphosis of visual systems in the sea lamprey. J. Gen. Physiol. 40, 901.
Discussion Dr Bensinger
In the Salvelinus speciesif the peak 503-nm rhodopsin differs from the 51%nm pigment by one amino acid, this could be detected by fingerprint analysis (peptide map). If only one spot difference is found, then only one substitution is likely. It could then be detected by eluting the different spots and quantified on an autoana,lyzer. Dr Bridges
We have plans roughly along those lines: there are three opsins to choosefrom in different speciesof the genus Salvelinus and at least two in Coregonus(these genera refer to the brook trout, lake trout, Arctic charr, whitefish, etc.). I feel that t,his
332
C’.
D.
IS.
BRIDGES
ASD
C.
E.
DELISLIS
approach, by letting Nature do half the work, will have a lot to tell us about, opsiit structure and the way in which the absorpt’ion properties of visual pigments art’ determined. Dr Robertson 11 000 years is a very short time on the evolutionary scale, I would think, judging from the work of Gillespie et al., that the presence of porphyropsin would be due to a molecule catalyzing the dehydrogenation of vitamin A, to vitamin A,. Gillespie has said that enzymes with extracellular substrates are more variable in their amino acid composition than are enzymes (proteins) which use int,racellular suhstrat.es. Dr Bridges Your point is very interesting. I agree that 11000 years is a very short time indeed, and that, is why we are so intrigued by the visual pigments of these various glacial relict populations around the world (cf. Bridges and Yoshikami, 1970a). However, as Mayr has pointed out, completion of the speciation process can occur in 105-lo6 years and the production of subspecies sometimes takes lo4 years or even less. In the isolates we have studied in the St Lawrence Lowlands there are so many possible points at, which the porphyropsin synthetic process may have become defective that we feel that it is a little early to speculate further. What we would like to do is to cross the pure rhodopsin race of smelt with one of its neighbors. That way we woultl see whether we have codominance, or whether we are dealing with dominant and recessive alleles.
Evolution of Visual Pigments C. D. B. BRIDGES* Department of Ophthalwmlqy, New York New York, N.Y. 10016, U.S.A.
Medical Ceder,
AND
C. E. Xoranda
DELISLE
ResearchCentre, Pointe Claire, Quebec,Canada
Rod visual pigments exhibit a variety of absorption maxima, determined partly by the nature of the prosthetic group, retinal or 3-dehydroretinal, and partly by the apoprotein opsin. Some animal taxa exhibit little diversity. On the other hand, selective pressures arising from the different light habitats of aquatic environments are believed to have in fish visual pigments, which often produced the correspondingly wide range of A,,, extends over 80 nm. Opsins appear to have similar molecular weights. Latest estimates of molecular weight suggest that they have approximately 315 aminoacidresidues. Some species, notably members of the salmon&e, have visual pigments with different A,,, yet have diverged only during the past several million years. This would indicate a very high rate of protein evolution in circumstances where we should expect that amino acid substitution would be severely restricted. Certain fish species (e.g. the deepwater sculpin, Myoxocephulw) have mixtures of visual pigments with A,,, separated by only 6 nm. The physiological usefulness of such a pair is dubious, but it may indicate polymorphism at the gene locus coding for opsin. The use of retinol and 3-dehydmntinof;-the basis of rhodopsin and porphyropsin, may vary between quite closely related species. Extraneous factors such as light and hormones are known t#o be important in amphibian larvae and fishes, but there is also clear evidence for genetic influences, as revealed by the divergent evolution of the system in various populations of originally anadromous smelt after only 10 660 years of isolat’ion.
1. Introduction The varied aquatic light environments that fishes occupy have clearly exerted evolutionary pressure on their visual pigments. More than 25 000 000 years ago the argentines and salmonidsdiverged from each other. Many of the former group became deep-seafishes and evolved visual pigments displaced towards the blue, so matching the blue-shifted spectrum of ambient light available for vision. Most of the salmonidae invaded fresh waters from the sea,either permanently or during spawning migrations, and hence developed red-shifted visual pigments corresponding to the reddish light that is often a feature of the freshwater environment (for review, seeBridges, 1972). The spectral location of a visual pigment absorption band can be influenced in two ways. One is to modify the apoprotein (opsin) while retaining the sameprosthetic group, viz. retinal in a rhodopsin or 3-dehydroretinal in a porphyropsin. Alternatively, rhodopsins and porphyropsins can be interchanged by substitution of one prosthetic group for the other, the apoprotein remaining unaltered. While many such changes have been accomplished by speciesevolving in a distant past under environmental conditions that are ill-defined from the standpoint of spectral light distribution, apparently somehave been more recent, occurring within the last few million years. The dominant influence of this period, which affected both speciation and distribution of many of the fishes discussedhere, was the advance and retreat of the great icesheetsduring the glacial epochs of the Pleistocene era, the last of which reached its maximum only 17 000-20 000 years ago.
2. Methods and Materials Collection
of material
Whole eyes or retinas were always obtained in dim red light from fish that) had been dark-adapted for 2-3 hr while still alive. Extracts of material from fish that were dead or nearly dead when placed in the dark frequently yielded some ,iso-pigment (9.cis prosthetic group) mixed with their natural visual pigment (the observations of Rotmans, Daemen and Bonting, 1972 and Putterman and Rollins, 1973 on regeneration of visual pigments may have some bearing on this finding). The ocular tissues were placed in stoppered vials, one for each fish (the retinas were immersed in &r/10 pH 7 phosphate buffer), sealed in light-tight containers and frozen on dry ice for transport to New York where they were transferred to a deep freeze until they were processed (usually within 1 week of collection). Extraction
procedure
Retinas were extracted according to the method of Bridges and Yoshikami (197Oa). Each pair of whole eyes was placed in a Petri dish containing 2.5 ml of McIlvaine’s pH 4% phosphate-citrate buffer and the choroid and retina scraped away from the sclera. After the sclera and lens had been picked out with forceps, the buffered suspension of the remaining tissues was pipetted into a 3-ml polypropylene centrifuge tube and centrifuged for 15 min at 17 000 rev/min in the 85-34 rotor of a Sorvall RC-2B centrifuge. This method was used because the retina became friable and extremely difficult to dissect out quantitatively after freezing and thawing. After centrifuging, the sedimented tissue was washed twice with distilled water then extracted with 2% digitonin (Merck reagent, thoroughly boiled before use to ensure maximum stability). The impurity of such extracts did not hinder the subsequent measurement of visual pigment composition. Similar results were obtained on occasions when comparatively pure preparations were made by first suspending the ground ocular material (without sclera and lens) in 37.5% sucrose, diluting, centrifuging and then washing several times with distilled water before extracting with digitonin. Analysis
and measurement
of viswll
pigment
composition
Visual pigments (in 2% digitonin solution containing 10 mM-hydroxylamine at pH 8.5) were identified by bleaching them in successive stages with monochromatic lights. The technique used was essentially that described by Dartnall (1952). The percentage of rhodopsin and porphyropsin was determined as outlined by Bridges and Yoshikami (1970b). A Zeiss PM& II spectrophotometer was used for measurement of absorption spectra, and bleaching was effected either by exposure to radiation from a high-intensity Bausch and Lomb monochromator in conjunction with suitable stray light filters, or to the non-isomerizing light from an incandescent lamp filtered through a yellow Corning glass no. 3-72. Liver “vitamins
A”
The methods used for analysis of the percentages of retinol and 3-dehydroretinol stored in the livers were identical with those described by Bridges and Yoshikami (1970a). A Cary 14 spectrophotometer was used to measure absorption spectra.
3. Results and Discussion Recent emergence of different
rhodopsins
In the salmonid fishes there changes that apparently involve
in the salmon&
are numerous examples of recent visual pigment the opsin moiety. Thus the rhodopsin of all Salmo
EVOLUTION
OF
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325
PIGMEN’TS
species examined (Munz and Beatty, 1965) has an absorption maximum at 503 nm and is quite distinct from that of Salve&us nama&ush, where the absorption maximum is at 511-512 nm (Munz and McFarland, 1965; Bridges and Delisle, 1974). It is considered unlikely that these genera diverged more than two million years ago (see Norden, 1961). Differences between rhodopsins of species within the genus Salve&us must, have occurred even more recently: the absorption maximum may be at, 51 l-512 nm for the lake charr (Salvelinus namaycush), at 508 nm in the Arct,ic charr and its derivatives (S. alp&us), or at 503 nm in the brook charr (S. fontinalisz Munz and McFarland, 1965; Bridges and Yoshikami, 1970a; Bridges and Delisle, unpublished). Similarly, while in the genus Coregonusthe rhodopsin is usually about 510 nm (Bridges and Yoshikami, 1970a) one speciesthat diverged in the British Isles no more than 20 000 years ago (Maitland, 1970) has a rhodopsin with absorption maximum shifted some 10 nm towards the red end of the spectrum (Bridges, 1965). How do such changescome about? Some evidence suggeststhat vertebrate visual pigments are homologous proteins (Heller, 1969) containing about 315 amino acid residues (a value based on the molecular weights of Robinson, Gordon-Walker and Bownds, 1972; Heitzman, 1972 and Daemen, de Grip and Jansen, 1972). Evolution by a.mino a.cid substitution in these proteins, however, would be severely restricted by the necessity for preserving certain essential features of the steric conformation at, the chromophore site (Bridges and Yoshikami, 1970a) and possibly by other considerations arising from the fact that rhodopsin is an important integral part of the photoreceptor outer segment membranes. The less rigorously defined fibrinopeptides show the highest mutation rates, amounting to 90 accepted point mutations or 52 observed differences per 100 amino acid residues-over 100 000 000 years (Dayhoff, 1972). In a visual pigment, this would mean a maximum observable difference between t’wo divergent lines of only 6.4 amino acids after 2 000 000 years. It is conceivable, of course, that new visual pigments could emerge quite rapidly if they were determined by a seriesof dominant and recessive alleles. In the salmonidae, however. codominance was established when S. namaycush x S. fontinabis hybrids were found to have mixtures of the parent pigments, the rhodopsins being separated by 8-9 nm (McFarland and Munz, 1965). Although such mixtures are extremely difficult to detect, in retinal extracts, in the following instance we document a thorough investigat)ion of the visual pigment from a glacial relict specieswhich we conclude possesses two rhodopsins separated by only 6-7 nm. Two closely si&ar rhodopsins ,in ~G4nE pigment evolutionZ
in the ret&a
of the deep-water
sculpin--rc
possible
stage
Illustrated in Fig. 1 are the absorption spectra from a retinal extract of the deepwater sculpin (Myoxocephalus quadricornis thompsonii) netted in a glacial lake in the Ottawa, valley, Quebec. It was first reported there by Delisle and van Vliet (1968). The absorption maximum is at 511 nm, and an initial red irradiation yielded a reckdisplaced difl’erence spectrum with maximum at 514-515 nm. This observation is often encountered in extracts which have mainly rhodopsin contaminated with a t*race of porphyropsin, such as those from Coreyonus &e&i. As shown in Fig. ‘L, however, in these casessubsequent difference spectra are identical with each other becausethe initial irradiation had creamed off the trace pigment. But in the caseof the seulpin, subsequent spectra shifted progressively as bleaching proceeded until a final value of 508-509 nm was reached. Even when the tirst irradiation bleached only 5%, of t,hetotal pigment (Fig. l), the corresponding difference spectra still had maxima
Wovelength
(nm)
FIN. 1. The problem of Myoxocep?dus visual pigments. The initial irradiation with X 660 nm light bleached only 5% of the total pigment: this was followed by irradiation at 650, 640 and 620 nm which left only 6% of the original pigment. This was then bleached by a final “yellow” (incandescent lamp filtered through Corning no. 3-72). In these experiments the difference spectra moved progressively 514-515 to 508-509 nm, as illustrated in Fig. 2. Temperature 25”C, hydroxylamine 10 mM, from h,,, pH 8.5, digitonin 2%.
c xl 100 P x t5 0” a
I
I
I
I,
400
I
III,,,,
I,,,
500 Wavelength
600 (nml
2. Difference spectra. (a) An example (from the retina of the whitefish Coregonus artedii) of a rhodopsin “contaminated” with a few per cent of porphyropsin. The initial red irradiation removes this pigment and produces a difference spectrum (dotted) that is displaced relatively to all subsequent ones (+ , x , A), which are identical with each other. These curves represent the bleaching of the purified rhodopsin, which has its absorption maximum at 510-511 nm. (b) The range of ourves obtained during the experiment in Fig. 1, where the visual pigment from Myoxocephalus retina was analyzed. In this case the spectra shift progressively from X,,, 5145 to 08 nm (. . ., the overall difference spectrum obtained by subtraction of the final fully bleached spectrum from the first). FIG.
EVOLUTION
OF
VISUAL
PIGMENTS
447
at 514-515 nm. Similarly, the shift was not due to a trace of blue-sensitive pigment, for a yellow irradiation following a red bleach that had removed all but 6% of the total pigment still produced a difference spectrum with maximum at 50&509 nm. The range of difference spectra obtained in a typical experiment on an extract from a single fish is illustrated in Fig. 2. Since all extracts were at 25°C and contained 10 m;\l-hydroxylamine it is considered very improbable that these observations are a,ttributable to photoproduct interference. Moreover, the initial spectra do not show the narrowing effect that would result from a red-displacement due to the formation of a substance with an absorption band overlapping that of the parent pigment. The most plausible conclusion, therefore, is that, these extracts contain roughly equal amounts of two rhodopsins with absorption maxima close t*o 514 and 508 nm respectively. The aqlvantage of two such pigments, in visual terms: is dubious. Since both pigments are present in individual specimens (although it is not known whether they are in the same retinal photoreceptors), it is possible that, this is evidence for gene duplication and subsequent mutation, perhaps after the species entered freshwaters from circumpolar seas a,t a time prior to the Wisconsin glaciation (McAllister, 1959). Post!yl&cd
evolution
of rhodopsin
ad
porphyropsi,l
i,~ isolated populations of thr
smelt (Osmeruseperlanus) We turn now to a casewhere in the course of evolutionary change the prosthet#ic group has been switched around. Because the porphyropsins as a group absorb at longer wavelengths than the rhodopsins they are more prevalent in freshwater fish retinas. Many diadromous speciessuch as the Pacific salmon and lamprey exchange their marine rhodopsin for porphyropsin at the onset of their spawning ascent into freshwat,ers (Crescitelli, 1956; Wald, 1957; Beatty, 1966). Consequently, it might be exprct’ed that if such a speciesbecame permanently landlocked. perhaps becauseof chaugesin climate and physiography of a region, there would be selective pressureto retain t’he red-sensibive pigment. In order to learn more about the recent evolution
100 miles
IJIG. 3. Sites where various smelt samples were taken (methods: scoop net, gill net or rod-and-line). (1) Rimouski: (2) St Lawrence, Rivibre Boyer; (3) St Lawrence, St Nicolas: (4) L. Mandeville; (5) L. Ouimet: (6) L. Heney; (7) L. Mesch; (8) L. Memphremagog; (9) L. Massawippi; (10) L. Utopia, Pl’ew Brunswick. Arrows indicate transplantations.
3’8
c’. D.
B.
BRIDGE,S
ASD
c’. E.
DELISLE
of this system, we have studied the distribution of rhodopsin and porphyrophin in different populations of the smelt, Osmerus eperlanus, a normally anadromous species that became landlocked in the St Lawrence Lowlands in Quebec during the retrea,t of the ice at the close of the last (Wisconsin) glacial epoch. Since isolation t,he migratory habit has persisted, perforce restricted to the ascent of small feeder streams. The locations of our various capture sites are marked on the map in Fig. 3. As might have been anticipated, the “free” smelt taken during their upstream spawning migration from the St Lawrence River (Osmerus eperlanus modax) a,nclt,he River Conway in the British Isles (Osmerus eperlanus e~erlanus) possessedvisual pigment composedalmost entirely of porphyropsin (absorption maximum 542 z 1 nm). The corresponding rhodopsin with absorption maximum at 512 nm predominated in non-spawners taken later in the year at site 1, near the St Lawrence estuary. Pure porphyropsin was also found in spawning smelt from the lake at site 4 (separated from the river by only 20 miles) and in Lake Utopia (site 10) near the Atlantic coast, where isolation occurred during upwarping of the landmass as the weight of glacier ice was removed. TABLE
1
Distribution of rhodopsin and porphyropsin in various spawning populations of the smelt (Osmerus eperlanus)
Source
L. Heney L. L. L. L. L. L. St R.
(6)
Comments
*
giants dwarfs
Utopia (10) Meach (7) Ouimet (5) Memphremagog (8) Maseawippi (9) Mandeville (4) Lawrence R. (R. Boyer) Conway (Gt. Britain)
Visual pigment composition i Mainly Mainly Gr0S.s porphyropsin rhodopsin mixture (more than (O-70% (more than porphyropsin) go(,):) 90%)
sympatrio
(2)
races
introd. introd.
from from
“free” “free”
fish fish
site 10 site 7
* Numbers in parenthesis refer to sites depicted on the map in Fig. 3. Two sites (1 and 3) were sources of non-spawning fish only. t Extracts were always prepared from individual fish. $ As in trout and rudd (Reuter, White and Wald, 1971; Muntz and Northmore, 1971), the dorsal half of the retina usually had more rhodopsin than the ventral, the maximum difference amounting to nearly 30%. The eyes of L. Massawippi specimens were not divided.
Contrary to expectation, however, it soon became apparent that smelt taken from other isolated lakes, although taxonomically indistinguishable from those discussed above, had very different visual pigment compositions. These are summarized in Table I. High proportions of rhodopsin were found in spawning fishesfrom sit,es8 and 9 on the southern side of the St Lawrence Lowlands. In fact, some individual specimens lacked porphyropsin altogether, although others ranged up to 60%. Over a period of
EVOLUTION
OF
VISUAL
PIGMENTS
329
3 years the average pigment composition of spawning fish at site 8 did not change significantly-there was 19% porphyropsin in 1971, 24% in 1972 and 26% in 1973. Porphyropsin had almost completely vanished from the eyes of smelt collected further westwards at site 6, in the Gatineau north of Ottawa. This lake supports a giant and dwarf race of smelt (Delisle and Veilleux, 1969), which are reproductively isola.ted by virtue of their different spawning times. In this instance, neither was found to have more than a few per cent of porphyropsin, either during spawning or at, any other time. Even the young had pure rhodopsin, irrespective of whether they were collected in summer a few months after hatching, or through holes in the ice at the end of the following winter. We then went on to examine fish from two other lakes (5 and 7) in the region, respectively north and east of site 6. Fifty years ago the lake at site 7 had been stocked by transplantation of fertilized eggs from Lake Utopia (lo), nearly 500 miles away. Subsequently, there was a second transplantation from site 7 into the lake at site 5. Both transplanted colonies differed from those of site 6 but were identical with the parent population in having nearly pure porphyropsin during spawning. From the point of view of light environment, these various lakes appear to differ littlta from each other (with the possible exception of the very shallow lake at site 4). so the blue-shifted visual sensitivity that would be associated with a predominantly rhodopsin system is not manifestly adaptive. What is the basis for these differences between smelt populations? Although vera high doses of exogenous preformed 3-dehydroretinol can effect a porphyropsin increase in kokanee salmon and rainbow trout (Beatty, 1972; Jaquest and Beatty, 1972), a converse effect attributable to lack of this precursor does not explain the situation in smelt. Thus the rhodopsin-rich site 6 group had as much as 500/, of their liver retinols in the form of 3-dehydroretinol: in contrast, the porphyropsin-rich group at site 5 had only 15%. The striking difference between the pigments of the autochthonous fish of site 6 and the transplanted populations of sites 5 and 7 suggest that local conditions such as feeding, predation and light-environment are probably not factors that determine visual pigment balance in the smelt, even though their importance in other species has been established (Dartnall, Lander and Munz, 1961: Bridges, 1965; Bridges and Yoshikami, 1970b; Allen, 1971). We conclude, therefore. that, we are dealing with different races of smelt where the ability to synthesize porphyropsin is genetically determined. Opsins do not appear to be selective for retinal or 3-dehydroretinal (cattle opsin will accept indiscriminately the correct &-isomers of retinal and 3-dehydroretinal to form t(he parent rhodopsin or its porphyropsin counterpart; Wald, 1953). Consequently. degeneration of the porphyropsin system may arise from failure of the ocular tissue?: t,o sequester preformed 3-dehydroretinol (originating in the liver) or from loss of any enzymes required specifically for utilization of 3-dehydroretinol. Possibilities include the ester hydrolase, alcohol dehydrogenase (unlikely) or the isomerizing system. Alt)ernatively, ocular 3-dehydroretinol may originate de novo by dehydrogenation of retinol, perhaps within the pigment epithelium* (cf. Ohtsu, Naito and Wilt, 1964; Naito and Wilt, 1962; see also Bridges and Yoshikami, 197Oc). Such a process would requirr a 3,4-dehydrogenase, or possibly a series of isoenzymes of varying efficacies * In this connection it is worth noting that the pigment epithelium of the goldfish contains onl) 3-dehydroretinol, even after massive injections of retinol (Bridges, unpublished), indicating that the tissrle accumulates 3.dehydroretinol preferentially (it is present in the liver) or that it dehydrogenates retinal before the latter reaches detectable proportions.
330
L’. D.
B.
BRIDGES
AKD
C. E.
DELISLE
(the salmonids typically maintain multiple molecular forms of many enz~n~t+~ probably the result of extensive gene duplication; Massaro, 1972). This would piovitle the genetic basis for individual variation. The time available for evolution of these different races of smelt is determined 1)~ the chronology of the Champlain Sea episode, a marine inundation of the St Lawrence Lowlands that followed in the wake of the retreating Wisconsin ice. The fishes of sites 6, 8 and 9 probably became isolated from the main gene pool of the “free” river smelt while the Sea was shoaling and freshening at its terminal Lampsilis La,ke phase. This would place the date between 11 000 and 8400 years ago (Prest. 1970). ACKNOWLEDGMENTS
This work was supported by PHS Research Grant no. 5 ROl EY 00461 from the National Eye Institute. Throughout the major period of the project, Dr Delisle was affiliated to the Aquarium de Mont&al. We gratefully of Dr D. D. Beatty and ,Mr Roy Bradford in collecting New Brunswick.
acknowledge the valuable assistance eyes from the smelt of Lake Ut,opia,
REFERENCES Allen,
D. M. (1971).
Photic
control
of the proportions
of two
visual
pigments
in a fish.
T;isior~ ties.
11, 1077. Beatty, D. D. (1966). A study of the succession of visual pigments in Pacific salmon (Oncorhyncus). Cad. J. Zool. 44,429. Beatty, D. D. (1972). Visual pigment changes in salmonid fishes in response to exogenous Lthyroxine, bovine TSH and 3-dehydroretinol. Vision Res. 12, 1947. Bridges, C. D. B. (1965). Absorption properties, interconversions and environmental adaptation of pigments from fish photoreceptors. Cold Spring Harbor Symp. Quant. Biol. 30,317. Bridges, C. D. B. (1972). The rhodopsin-porphyropsin visual system. In Handbook of Sensory Physiology, vol. viii (l), pp. 417480. Springer-Verlag, Berlin. Bridges, C. D. B. and Delisle, C. E. (1974). Brief observations concerning the visual pigments of some selected fishes from Lake Heney, Quebec, a relict of glacial Lake Gatineau. Vision Res. 14, 187. Bridges, C. D. B. and Yoshikami, S. (1970a). Distribution and evolution of visual pigments in salmonid fishes. Vision Res. 10, 609. Bridges, C. D. B. and Yoshikami, S. (197Ob). The rhodopsin-porphyropsin system in freshwater fishes 1. Effects of age and photic environment. Vision Res. 10, 1315. Bridges, C. D. B. and Yoshikami, S. (197Oc). The rhodopsit-porphyropsin in freshwater fishes 2. Turnover and interconversion of visual pigment prosthetic groups in light and darknessrole of the pigment epithelium. r&ion Res. 10, 1333. Crescitelli, F. (1956). The nature of the lamprey visual pigment. J. Gen. Physiol. 39,423. Daemen, F. J. M., De Grip, W. J. and Jansen, P. A. A. (1972). Biochemical aspects of bhe visual process XX. The molecular weight of rhodopsin. Biochim. Biophys. Acta 271,419. Dartnall, H. J. A. (1952). Visual pigment 467, a photosensitive pigment present in tenth retinae. J. Physiol. 116,257. Dartnall, H. J. A., Lander, M. R. and Munz, F. W. (1961). Periodic changes in the visual pigment of a fish. In Progress in Photobiology (ed. Christensen, B. and Buchmann, B.). Pp. 203-213. Elsevier, Amsterdam. Dayhoff, M. 0. (1972). Atlas of protein sequence and structure. National Biomedical Res. Foundation, Washington, D.C. Delisle, C. and Van Vliet, W. (1968). First records of the sculpins Myoxocephalus thompsonii and Cottus ricei from the Ottawa valley, southwestern Quebec. J. Fish. Res. Bd. Canada 25,273;~. Delisle, C. and Veilleux, C. (1969). R&partition geographique de l’eperlan arc-en-ciel Osnrerus eperlanus mcrdax et de Glugea hertwigi (sporozoa: microsporidia) en eau deuce, au Qn6ben. Natural&e Can. 96, 337. Futterman, S. and Rollins, M. H. (1973). Evidence for the involvement of a reduced flavin isomerization catalyst in the regeneration of bleached rhodopsin. Invest. Ophthalmol. 12, 234.
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Heitzman, H. (1972). Rhodopsin is the predominant protein of rod outer segment membranes. Nature New Biol. 235, 114. Reller, J. (1969). Comparative study of a membrane protein. Characterization of bovine, rat and frog visual pigments. Biochem. 8, 675. Jaquest, W. L. and Beatty, D. D. (1972). Visual pigment changes in the rainbow trout, S&LO guirdnerii. Canad. J. Zool. 50, 1117. Maitland, P. S. (1970). The origin and present dist,ribution of Coregonus in the British Isles. In Biology of Coregonid Fishes (Ed. Lindsey, C. C. and Woods, C. 8.). Pp. 99-114. University of Manitoba Press, Winnipeg. Massaro, E. J. (1972). Isozyme patterns of coregonine fishes: evidence for multiple cistrons for lactate and malate dehydrogenases and achromatic bands in the tissues of Prosobium cylindraceum (Pallas) and P. coulteri (Eigenmann and Eigenmann). J. Exp. Zool. 179, 247. Mc,4llister, D. E. (1959). The origin andstatus of the deepwater sculpin. iWyoxocepha,Zua thompsonii. a Nearctic glacial relict. Bull. Nat. Mus. Canada 172, 44. McFarland, W. N. and Munz, F. W. (1965). Codominance of visual pigments in hybrid fishes. Science (New York) 150, 1055. Muntz, W. R. A. and Northmore, D. P. M. (1971). Visual pigments from different parts of the retina in rudd and trout. Vision Res. 11, 551. Munz. F. W. and Beatty, D. D. (1965). A critical analysis of the visual pigments of salmon and tsout. Vision Res. 5, 1. Munz, F. W. and McFarland, W. N. (1965). B suggested hereditary mechanism for visual pigments of chars (Salvelinus spp.). Nature (London) 206,955. Naito, K. and Wilt, F. H. (1962). The conversion of vitamin A, to retinene, in a freshwat,er fish. J. Biol. Chem. 231, 3060. Norden, C. R. (1961). Comparative osteology of representative salmonid fishes, with particular reference to the grayling (Thyrnallus arcticus) and its phylogeny. J. Fish. Res. Bd. Cnnndn 18, 679. Ohtsu, K., Naito, K. and Wilt, F. H. (1964). Metabolic basis of visual pigment conversion in metamorphosing Rana catesbiana. Develop. Biol. 10, 216. Prest . V. K. (1970). Quaternary geology of Canada. In Geology and Economic Xinera.1.r of Canadrr, 5th ed. Econ. Rep. 1, chap. xii, pp. 676-725. Department’ Energy, Mines and Resources. Ottawa. Reuter, T. E., Whit,e, R’. H. and Wald, CT, (1971). Rhodopsin and porphyropsin fields in the adult, bullfrog retina. J. Gen. Physiol. 58, 351. Robinson, W. E., Gordon-Walker, A. and Bownds, D. (1972). Molecular weight of frog rhodopsin. Xature New Bill. 235, 112. Rotmans, J. P., Daemen, F. J. M. and Bonting, S. L. (1972). Biochemical aspects of the visual process XIX. Formation of isorhodopsin from photolyzed rhodopsin by bacterial action. Biochim. Biophys. Acta 267,583. Wald, G. (1953). Vision. Fed. Proc. 12, 606. Wald, G. (1957). The metamorphosis of visual systems in the sea lamprey. J. Gen. Physiol. 40, 901.
Discussion Dr Bensinger
In the Salvelinus speciesif the peak 503-nm rhodopsin differs from the 51%nm pigment by one amino acid, this could be detected by fingerprint analysis (peptide map). If only one spot difference is found, then only one substitution is likely. It could then be detected by eluting the different spots and quantified on an autoana,lyzer. Dr Bridges
We have plans roughly along those lines: there are three opsins to choosefrom in different speciesof the genus Salvelinus and at least two in Coregonus(these genera refer to the brook trout, lake trout, Arctic charr, whitefish, etc.). I feel that t,his
332
C’.
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IS.
BRIDGES
ASD
C.
E.
DELISLIS
approach, by letting Nature do half the work, will have a lot to tell us about, opsiit structure and the way in which the absorpt’ion properties of visual pigments art’ determined. Dr Robertson 11 000 years is a very short time on the evolutionary scale, I would think, judging from the work of Gillespie et al., that the presence of porphyropsin would be due to a molecule catalyzing the dehydrogenation of vitamin A, to vitamin A,. Gillespie has said that enzymes with extracellular substrates are more variable in their amino acid composition than are enzymes (proteins) which use int,racellular suhstrat.es. Dr Bridges Your point is very interesting. I agree that 11000 years is a very short time indeed, and that, is why we are so intrigued by the visual pigments of these various glacial relict populations around the world (cf. Bridges and Yoshikami, 1970a). However, as Mayr has pointed out, completion of the speciation process can occur in 105-lo6 years and the production of subspecies sometimes takes lo4 years or even less. In the isolates we have studied in the St Lawrence Lowlands there are so many possible points at, which the porphyropsin synthetic process may have become defective that we feel that it is a little early to speculate further. What we would like to do is to cross the pure rhodopsin race of smelt with one of its neighbors. That way we woultl see whether we have codominance, or whether we are dealing with dominant and recessive alleles.